Cold Atmospheric Plasma Attenuates Breast Cancer Cell Growth Through Regulation of Cell Microenvironment Effectors

Breast cancer exists in multiple subtypes some of which still lack a targeted and effective therapy. Cold atmospheric plasma (CAP) has been proposed as an emerging anti-cancer treatment modality. In this study, we investigated the effects of direct and indirect CAP treatment driven by the advantageous nanosecond pulsed discharge on breast cancer cells of different malignant phenotypes and estrogen receptor (ER) status, a major factor in the prognosis and therapeutic management of breast cancer. The main CAP reactive species in liquid (i.e. H2O2, NO2−/NO3−) and gas phase were determined as a function of plasma operational parameters (i.e. treatment time, pulse voltage and frequency), while pre-treatment with the ROS scavenger NAC revealed the impact of ROS in the treatment. CAP treatment induced intense phenotypic changes and apoptosis in both ER+ and ER- cells, which is associated with the mitochondrial pathway as evidenced by the increased Bax/Bcl-2 ratio and cleavage of PARP-1. Interestingly, CAP significantly reduced CD44 protein expression (a major cancer stem cell marker and matrix receptor), while differentially affected the expression of proteases and inflammatory mediators. Collectively, the findings of the present study suggest that CAP suppresses breast cancer cell growth and regulates several effectors of the tumor microenvironment and thus it could represent an efficient therapeutic approach for distinct breast cancer subtypes.

rs62139665 Polymorphism in the Promoter Region of EpCAM Is Associated With Hepatitis C Virus-Related Hepatocellular Carcinoma Risk in Egyptians

Hepatocellular carcinoma (HCC) is a universal health problem that is particularly alarming in Egypt. The major risk factor for HCC is hepatitis C virus (HCV) infection which is a main burden in Egypt. The epithelial cell adhesion molecule (EpCAM) is a stem cell marker involved in the tumorigenesis and progression of many malignancies, including HCC. We investigated the association of -935 C/G single nucleotide polymorphism in EpCAM promoter region (rs62139665) with HCC risk, EpCAM expression and overall survival in Egyptians. A total of 266 patients (128 HCV and 138 HCC cases) and 117 age- and sex-matched controls participated in this study. Genotyping, performed using allelic discrimination and confirmed by sequencing, revealed a significant association between EpCAM rs62139665 and HCC susceptibility, with higher GG genotype and G allele distribution in HCC patients than in non-HCC subjects. Such association was not detected in HCV patients compared to controls. EpCAM gene expression levels, determined in blood by RT-qPCR, and its serum protein expression levels, determined by ELISA, were significantly higher in GG relative to GC+CC genotype carriers in HCV and HCC patients in a recessive model. ROC analysis of EpCAM protein levels revealed significant discriminatory power between HCC patients and non-HCC subjects, with improved diagnostic accuracy when combining α-fetoprotein and EpCAM compared to that of α-fetoprotein alone. Altogether, EpCAM rs62139665 polymorphism is significantly associated with HCC and with EpCAM gene and protein expression levels in the Egyptian population. Moreover, serum EpCAM levels may hold promise for HCC diagnosis and for improving the diagnostic accuracy of α-fetoprotein.

miR-671-5p Inhibition by MSI1 Promotes Glioblastoma Tumorigenesis via Radioresistance, Tumor Motility and Cancer Stem-like Cell Properties

MicroRNAs (miRNAs) could be potential biomarkers for glioblastoma multiforme (GBM) prognosis and response to therapeutic agents. We previously demonstrated that the cancer stem cell marker Musashi-1 (MSI1) is an RNA binding protein that promotes radioresistance by increasing downstream RNA stability. To identify that MSI1 interacts with miRNAs and attenuates their function, we also get candidate miRNAs from the mRNA seq by predicting with TargetScan software. miR-671-5p in GBM cells interacts with MSI1 by intersecting the precipitated miRNAs with the predicted miRNAs. Notably, overexpression of MSI1 reversed the inhibitory effect of miR-671-5p. The phenotype of miR-671-5p in GBM cells could affect radiosensitivity by modulating the posttranscriptional activity of STAT3. In addition, miR-671-5p could attenuate tumor migration and cancer stem cell (CSC) characteristics by repressing the posttranscriptional activity of TRAF2. MSI1 may regulate GBM radioresistance, CSCs and tumor motility through miR-671-5p inhibition to increasing STAT3 and TRAF2 presentation. In vivo, the GBM tumor size was inversely correlated with miR-671-5p expression, but tumorigenesis was promoted by STAT3 and TRAF2 activation in the miR-671-5p-positive GBM population. miR-671-5p could be activated as a novel therapeutic target for GBM and has potential application as a predictive biomarker of glioblastoma prognosis.

Flower meristem maintenance by TILLERS ABSENT 1 is essential for ovule development in rice

ABSTRACT Plant development depends on the activity of pluripotent stem cells in meristems, such as the shoot apical meristem and the flower meristem. In Arabidopsis thaliana, WUSCHEL (WUS) is essential for stem cell homeostasis in meristems and integument differentiation in ovule development. In rice (Oryza sativa), the WUS ortholog TILLERS ABSENT 1 (TAB1) promotes stem cell fate in axillary meristem development, but its function is unrelated to shoot apical meristem maintenance in vegetative development. In this study, we examined the role of TAB1 in flower development. The ovule, which originates directly from the flower meristem, failed to differentiate in tab1 mutants, suggesting that TAB1 is required for ovule formation. Expression of a stem cell marker was completely absent in the flower meristem at the ovule initiation stage, indicating that TAB1 is essential for stem cell maintenance in the ‘final’ flower meristem. The ovule defect in tab1 was partially rescued by floral organ number 2 mutation, which causes overproliferation of stem cells. Collectively, it is likely that TAB1 promotes ovule formation by maintaining stem cells at a later stage of flower development.

Transcriptomic heterogeneity of Sox2-expressing pituitary cells

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells (HPC), nonhormonal folliculostellate cells (FSC) and pituicytes, vascular pericytes and endothelial cells, and putative Sox2-expressing stem cells. Here, we used scRNAseq analysis of adult female rat pituitary cells to study the heterogeneity of pituitary cells with a focus on evaluating the transcriptomic profile of the Sox2-expressing population. Samples containing whole pituitary and separated anterior and posterior lobe cells allowed the identification of all expected pituitary resident cell types and lobe-specific subpopulations of vascular cells. Sox2 was expressed uniformly in all FSC, pituicytes, and a fraction of HPC. FSC comprised two subclusters; FSC1 contained more cells but expressed less genetic diversity compared to FSC2. The latter contained proliferative cells, expressed genes consistent with stem cell niche formation, including tight junctions, and shared genes with HPC. The FSC2 transcriptome profile was also consistent with the activity of pathways regulating cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. The expression of other stem cell marker genes was common for FSC and pituicytes (Sox9, Cd9, Hes1, Vim, S100b) or cell type-specific (FSC: Prop1, Prrx1, Pitx1, Pitx2, Lhx3; pituicytes: Fgf10, Tbx3, Lhx2, Nkx2-1, Rax). FSC and pituicytes also expressed other astroglial marker genes, some common and other distinct, consistent with their identities as astroglial cells of the pituitary. These data suggest functional heterogeneity of FSC, with a larger fraction representing classical FSC, and a smaller fraction containing active stem-like cells and HPC-committed progenitors.

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma

We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm’s canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm’s canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.

A schizophrenia subgroup with elevated inflammation displays reduced microglia, increased peripheral immune cell and altered neurogenesis marker gene expression in the subependymal zone

Inflammation regulates neurogenesis, and the brains of patients with schizophrenia and bipolar disorder have reduced expression of neurogenesis markers in the subependymal zone (SEZ), the birthplace of inhibitory interneurons. Inflammation is associated with cortical interneuron deficits, but the relationship between inflammation and reduced neurogenesis in schizophrenia and bipolar disorder remains unexplored. Therefore, we investigated inflammation in the SEZ by defining those with low and high levels of inflammation using cluster analysis of IL6, IL6R, IL1R1 and SERPINA3 gene expression in 32 controls, 32 schizophrenia and 29 bipolar disorder cases. We then determined whether mRNAs for markers of glia, immune cells and neurogenesis varied with inflammation. A significantly greater proportion of schizophrenia (37%) and bipolar disorder cases (32%) were in high inflammation subgroups compared to controls (10%, p < 0.05). Across the high inflammation subgroups of psychiatric disorders, mRNAs of markers for phagocytic microglia were reduced (P2RY12, P2RY13), while mRNAs of markers for perivascular macrophages (CD163), pro-inflammatory macrophages (CD64), monocytes (CD14), natural killer cells (FCGR3A) and adhesion molecules (ICAM1) were increased. Specific to high inflammation schizophrenia, quiescent stem cell marker mRNA (GFAPD) was reduced, whereas neuronal progenitor (ASCL1) and immature neuron marker mRNAs (DCX) were decreased compared to low inflammation control and schizophrenia subgroups. Thus, a heightened state of inflammation may dampen microglial response and recruit peripheral immune cells in psychiatric disorders. The findings elucidate differential neurogenic responses to inflammation within psychiatric disorders and highlight that inflammation may impair neuronal differentiation in the SEZ in schizophrenia.

Ascorbic Acid/Retinol and/or Inflammatory Stimuli’s Effect on Proliferation/Differentiation Properties and Transcriptomics of Gingival Stem/Progenitor Cells

The present study explored the effects of ascorbic-acid (AA)/retinol and timed inflammation on the stemness, the regenerative potential, and the transcriptomics profile of gingival mesenchymal stem/progenitor cells’ (G-MSCs). STRO-1 (mesenchymal stem cell marker) immuno-magnetically sorted G-MSCs were cultured in basic medium (control group), in basic medium with IL-1β (1 ng/mL), TNF-α (10 ng/mL) and IFN-γ (100 ng/mL, inflammatory-medium), in basic medium with AA (250 µmol/L) and retinol (20 µmol/L) (AA/retinol group) or in inflammatory medium with AA/retinol (inflammatory/AA/retinol group; n = 5/group). The intracellular levels of phosphorylated and total β-Catenin at 1 h, the expression of stemness genes over 7 days, the number of colony-forming units (CFUs) as well as the cellular proliferation aptitude over 14 days, and the G-MSCs’ multilineage differentiation potential were assessed. Next-generation sequencing was undertaken to elaborate on up-/downregulated genes and altered intracellular pathways. G-MSCs demonstrated all mesenchymal stem/progenitor cells characteristics. Controlled inflammation with AA/retinol significantly elevated NANOG (p < 0.05). The AA/retinol-mediated reduction in intracellular phosphorylated β-Catenin was restored through the effect of controlled inflammation (p < 0.05). Cellular proliferation was highest in the AA/retinol group (p < 0.05). AA/retinol counteracted the inflammation-mediated reduction in G-MSCs’ clonogenic ability and CFUs. Amplified chondrogenic differentiation was observed in the inflammatory/AA/retinol group. At 1 and 3 days, the differentially expressed genes were associated with development, proliferation, and migration (FOS, EGR1, SGK1, CXCL5, SIPA1L2, TFPI2, KRATP1-5), survival (EGR1, SGK1, TMEM132A), differentiation and mineral absorption (FOS, EGR1, MT1E, KRTAP1-5, ASNS, PSAT1), inflammation and MHC-II antigen processing (PER1, CTSS, CD74) and intracellular pathway activation (FKBP5, ZNF404). Less as well as more genes were activated the longer the G-MSCs remained in the inflammatory medium or AA/retinol, respectively. Combined, current results point at possibly interesting interactions between controlled inflammation or AA/retinol affecting stemness, proliferation, and differentiation attributes of G-MSCs.

Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.